摘要
A new fibrinogenase(EC 3.4.2 I.5)was isolated and purified from the venom of Chinese habu snake(Trimeresurus研ucrosg“ama似s)by DEAE—SephadexA-50,DEAE—Sepharose CL一6B,MonoQ(FPLC)CG.1umn chromatography.It showed a single protein band both in sodium dodeeyl sulfate(SDS)·polyacrylami-de geI electrophoresis and alkaline polyacrylamide gel electrophoresis.The molecular weight was estimated tobe 26000 by SDS·p01yacrylam|de gel electrophoresis.The isoeleetric point was found to be pH 4.7.Itwas a glycoprotein containing 6.4‘%carbohydrate with o.3%neutral sugar,1.2%sialic acid,4.9%he.xosamine.It was composed of about 1 78 amino acid residues and rich in glycine and aspartic acid.Thefibrinogenase of the venom of T.munro$quclmatu$TWV№was heat stable but labile to acid.Its extinctioncoefficient(1mg/m1)at 280rim was 1.558.Purified TMVFg had strong arginine esterase activity·the Kmto benzoylarginine ethylester(BAEE)was 1.4×1 0一M.The enzyme activity could be inhibited by pheny.1mefha口esulfonyfluoride(PMSF),but was not affected by ethylenediamine tetraacetlc acid(EDTA).TMVFghad fibrinogenolytie activityl electrophoresis of fibrinogen degraded with TMVFg revealed the rapiddisappearance of the口(alpba)and B(beta)‘chains and the appearance I】f lower molecular weight frag.merits.TMVFg did not cause fibrinogen solution clotting,nor coagulating plasma and showed n^ither hemorr-hagic activity nor proteolytic activity toward casein.TMVFg had activati^lg fibrinolytic activity
通过DEAE-SephadexA-50,DEAE-SepharoseCL-6B,MonoQ(FPLC)三步离子交换柱层析,纯化得到一新的纤维蛋白原溶酶,在碱性聚丙烯酰胺凝胶电泳和SDS-聚丙烯酰胺凝胶电泳上均呈单一的蛋白带。分子量为26000,等电点pI4.7;它是一个糖蛋白,含糖量6.4％,其中中性糖0.3％,己糖胺4.9％,唾液酸1.2％。烙铁头蛇毒纤维蛋白原溶酶TMVFg由187个氨基酸组成,含有较高的酸性氨基酸,此外甘氨酸含量也较高。TMVFg热稳定性强,而酸不稳定,在280nm处具有典型的蛋白吸收峰,在无离子水中紫外消光系数E0.1％/280nm=1.558。纯化的TMVFS具有较强的精氨酸酯酶活力,对苯甲酰-L-精氨酸乙酯(BAEE)的Km值为1.4×10^(-3)M。TMVFS的活性受苯甲基丹磺酰氟(PMSF)抑制,乙二胺四乙酸(EDTA)对活性无影响。TMVFg不能使纤维蛋白原凝固,但能水解纤维蛋白原α、β链。纤溶实验表明TMVFg具有激活纤溶作用。纯化的烙铁头蛇毒纤维蛋白原溶酶对酪蛋白无作用,无出血活性,因而与凝血酶样酶、出血毒素及纤维蛋白原溶酶(OUYANG et al,1977) 明显不同。
